产气荚膜梭菌新双组份信号转导系统的筛选及其分子调控机制研究

Clostridium perfringens can cause animal necrotic enteritis, gas gangrene and food poisoning. Two-component signal transduction system regulates multiple biological functions of bacteria. At present, there is no report on two-component signal transduction systems of Clostridium perfringens in domestic, although there were some research works carried out in overseas, there is no report on excavating new two-component signal transduction systems and the research of its regulatory mechanism based on total genes and proteins. Therefore, on the basis of previous studies, the genome sequencing of Clostridium perfringens isolate strain will be revealed in this project and the new two-component signal transduction system will be explored.CRISPR/Cas9 and homologous recombination technology will be used to construct the gene deletion strains of new two-component signal transduction system of Clostridium perfringens. RNA-seq and iTraq will used to obtain the genes and proteins regulated by the new two-component signal transduction system in the deletion and wild strains, and the function of the genes will be verified by Real-Time PCR. Analyzing genes and proteins which were regulated by new two-component signal transduction systems by bioinformatics methods, reveal molecular regulation mechanism of new two-component signal transduction system, all of the research will provide the theoretical basis for the pathogenesis of Clostridium perfringens, and provide scientific basis for the development of new drugs and vaccine.

产气荚膜梭菌能引起动物坏死性肠炎、气性坏疽和食物中毒。双组份信号转导系统调控细菌多种生物学功能。目前关于产气荚膜梭菌双组份信号转导系统的研究国内未见报道，国外虽开展了一些工作，但未见从全基因组角度发掘新双组份信号转导系统，也未见从全基因和全菌蛋白角度对新双组份信号转导系统调控机制进行研究的报道。因此，本项目将在前期研究基础之上对产气荚膜梭菌分离株进行基因组测序，发掘新的双组份信号转导系统。用CRISPR/Cas9和同源重组技术构建产气荚膜梭菌新双组份信号转导系统基因缺失菌株。用RNA-seq和iTraq技术对缺失株与野生株进行研究，获得新双组份信号转导系统调控的基因与蛋白，Real-Time PCR验证功能基因。生物信息学方法分析新双组份信号转导系统调控的基因与蛋白，全面揭示新双组份信号转导系统分子调控机制，这将为产气荚膜梭菌致病机理提供基础理论，还可为该菌新药和新型疫苗研制提供科学依据。

产气荚膜梭菌是引起各种动物坏死性肠炎、肠毒血症和人畜创伤性气性坏疽和人类食物中毒的主要病原菌之一。双组份信号转导系统是细菌体内一类响应外界信号变化的调控系统，调控细菌致病性、外界环境适应性、耐药性、生物被膜形成、毒力表达等多种重要的生物学功能。因此，深入研究细菌双组份信号转导系统对于阐明病原菌分子致病机理、研发新型抗菌药物和基因工程疫苗具有重要意义。本项目通过对产气荚膜梭菌进行全基因组测序与分析，筛选产气荚膜梭菌双组份信号转导系统。构建产气荚膜梭菌双组份信号转导系统基因缺失菌株，采用RNA-seq和iTraq技术对缺失株与野生株进行差异表达基因与蛋白的分析，以期为产气荚膜梭菌双组份信号转导系统分子调控机制的深入研究提供理论基础。. 本项目通过全基因组测序技术对产气荚膜梭菌进行测序与分析，获得40个双组份信号基因。通过用CRISPR/Cas9和同源重组技术构建产气荚膜梭菌yesN双组份信号基因缺失菌株（CPAΔyesN）。CPAΔyesN缺失株与CPA野生株生物学特性比较结果表明，缺失株CPAΔyesN相较于野生株CPA，htrA、rpoA、rpoN、filC等生物被膜基因mRNA水平均呈现下调表达，说明yesN双组份信号基因的缺失下调生物被膜基因的表达，进而影响产气荚膜梭菌生物被膜的形成。缺失株CPA ΔyesN相较于野生株CPA，α毒素编码基因mRNA水平呈现下调表达，说明yesN双组份信号基因的缺失下调了产气荚膜梭菌α毒素的表达。转录组测序结果表明，缺失株CPA ΔyesN相较于野生株CPA共有901个差异表达基因，其中上调表达基因273个，下调表达基因628个。GO富集结果表明，差异基因富集在1856个GO功能类别中。KEGG富集结果表明，差异表达基因富集在72个KEGG通路中，上调的315条差异基因主要涉及53个信号通路，下调的553条差异基因主要涉及65个信号通路。蛋白质组学iTraq结果表明，缺失株CPA ΔyesN相较于野生株CPA共有1570个差异表达蛋白，其中显著上调的差异蛋白794个，显著下调的差异蛋白776个。GO 富集结果表明，差异蛋白富集在1123个GO功能类别中。KEGG富集结果表明，差异表达蛋白富集到了57个通路中。